Gilles M. Leclerc

AMPK-induced activation of Akt by AICAR is mediated by IGF-1R dependent and independent mechanisms in acute lymphoblastic leukemia

Background Children with Acute Lymphoblastic Leukemia (ALL) diagnosed with resistant phenotypes and those who relapse have a dismal prognosis for cure. In search for novel treatment strategies, we identified the AMP activated protein kinase (AMPK) as a potential drug target based on its effects on cell growth and survival. We have shown previously that AICAR-induced AMPK activation also induced a compensatory survival mechanism via PI3K/Akt signaling. Results In the present study, we further investigated the downstream signaling induced by AMPK activation in ALL cells. We found that AICAR-induced AMPK activation resulted in up-regulation of P-Akt (Ser473 and Thr308) and decrease of P-mTOR (Ser2448) expression and downstream signaling. We determined that activation of P-Akt (Thr308) was mediated by AMPK-induced IGF-1R activation via phosphorylation of the insulin receptor substrate-1 (IRS-1) at Ser794. Inhibition of IGF-1R signaling using the tyrosine kinase inhibitor HNMPA(AM)3 resulted in significant decrease in P-IRS-1 (Ser794) and P-Akt (Thr308). Co-treatment of AICAR plus HNMPA(AM)3 prevented AMPK-induced up-regulation of P-Akt (Thr308) but did not alter the activation of P-Akt (Ser473). Inhibition of AMPK using compound-C resulted in decreased P-Akt expression at both residues, suggesting a central role for AMPK in Akt activation. In addition, inhibition of IGF-1R signaling in ALL cells resulted in cell growth arrest and apoptosis. Additional Western blots revealed that P-IGF-1R (Tyr1131) and P-IRS-1 (Ser794) levels were higher in NALM6 (Bp-ALL) than CEM (T-ALL), and found differences in IGF-1R signaling within Bp-ALL cell line models NALM6, REH (TEL-AML1, [t(12;21)]), and SupB15 (BCR-ABL, [t(9;22)]). In these models, higher sensitivity to IGF-1R inhibitors correlated with increased levels of IGF-1R expression. Combined therapy simultaneously targeting IGF-1R, AMPK, Akt, and mTOR pathways resulted in synergistic growth inhibition and cell death. Conclusions Our study demonstrates that AMPK activates Akt through IGF-1R dependent and independent mechanisms. Co-targeting IGF-1R and related downstream metabolic and oncogenic signaling pathways represent a potential strategy for future translation into novel ALL therapies.

AMPK and Akt Determine Apoptotic Cell Death following Perturbations of One-Carbon Metabolism by Regulating ER Stress in Acute Lymphoblastic Leukemia

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates [methotrexate (MTX) or pemetrexed] has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein show that sustained endoplasmic reticulum (ER) stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further upregulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also show that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical proapoptotic versus prosurvival effects of AMPK activation. Mol Cancer Ther; 10(3); 437–47. ©2011 AACR.

Metformin Induces Apoptosis through AMPK-Dependent Inhibition of UPR Signaling in ALL Lymphoblasts

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin’s cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL.

Inhibition of Akt Potentiates 2-DG–Induced Apoptosis via Downregulation of UPR in Acute Lymphoblastic Leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DGs cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

Histone deacetylase inhibitors induce FPGS mRNA expression and intracellular accumulation of long-chain methotrexate polyglutamates in childhood acute lymphoblastic leukemia: implications for combination therapy

Children with acute lymphoblastic leukemia (ALL) diagnosed with resistant phenotypes, and those who relapse, have a dismal prognosis for cure. The antifolate methotrexate (MTX), a universal component of ALL therapies, is metabolized by folylpoly-γ-glutamate synthetase (FPGS) into long-chain polyglutamates (MTX-PG3−7), resulting in enhanced cytotoxicity from prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). Using DNaseI assays, we identified a hypersensitive site upstream from exon-1, suggesting chromatin remodeling could alter FPGS expression. We demonstrated that histone deacetylase-1 (HDAC1) is recruited by NFY and Sp1 transcription factors to the FPGS promoter in ALL cell lines. We examined the effect of histone deacetylase inhibitors (HDACIs) sodium butyrate and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes. HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas DHFR and TS mRNA expression was decreased. Combination treatment with MTX plus SAHA significantly increased cytotoxicity and apoptosis in B- and T-ALL cell lines as compared with each drug alone (CI⩽0.8). SAHA increased the intracellular accumulation of long-chain MTX-PG3−7. Therefore, HDACI-induced FPGS expression increases the accumulation of MTX-PG3−7 and cytotoxicity in ALL cell lines, which is potentiated by DHFR and TS downregulation. The synergism exhibited by the combination of MTX and SAHA warrants clinical testing in ALL patients.

Administration of connexin43 siRNA abolishes secretory pulse synchronization in GnRH clonal cell populations

GnRH is released from hypothalamic neurons in coordinated pulses, but the cellular basis for this process is poorly understood. Previously, we found that secretory pulses from GT1-7 cells became synchronized with time in culture. Using this culture model, we investigated whether the gap junction proteins connexin43 (Cx43) and/or connexin26 (Cx26) are involved in this synchronization. Our results reveal that cytoplasmic densities immunoreactive for Cx43, and mRNA or protein for Cx43 increase with time in culture. Also, microinjection of day-3 cultures with siRNA for Cx43 abolished synchronized activity at day 7. Interestingly, cytoplasmic plaques, mRNA, or protein for Cx26 remained stable with culture time and Cx26 siRNA administration did not alter secretory activity. Our findings demonstrate that Cx43, but not Cx26 is necessary for synchronized secretory activity in these GT1-7 cultures and raise the possibility that Cx43-related gap junctions may be important in GnRH neuronal coordination in the hypothalamus.

Abstract 605: A novel role for AMPK as a molecular switch regulating the activation of Akt and RAS signaling in acute lymphoblastic leukemia

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we identified AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 and CCRF-CEM cells with the AMPK activators AICAR and metformin induced significant cell growth inhibition and apoptosis. Rescue experiments using the AMPK inhibitor compound-C (CC) failed to completely abrogate the cytotoxic effects induced by AICAR, attenuating AICARs apoptosis by 42% in CCRF-CEM and 45% in NALM6. Whereas when used alone, both CC and Ara-A induced significant apoptosis in ALL cells. Using the caspase inhibitors Z-VAD and Z-IETD, we demonstrated that activation of AMPK by AICAR induced cell death via both caspase-dependent and independent mechanisms, whereas inhibition of AMPK by CC induced apoptosis mainly via the intrinsic caspase-dependent pathway. To examine the effects of AMPK activation vs. inhibition on downstream signaling, we used Western blot analysis of key signaling factors associated with the PI3K/Akt/mTOR and RAS/RAF/ERK pathways in ALL cells. We found that AICAR and CC exerted opposite effects on PI3K/Akt and RAS pathway signaling. CC decreased P-Akt (Ser473) and activated the RAS pathway, whereas AICAR increased P-Akt, suggesting that AMPK may play a role as regulator of Akt and RAS activity. We showed that activation of AMPK and Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF at Ser621 and Ser259, respectively. Inhibition of the RAS pathway with farnesylthiosalicylic acid (FTS) and U-0126 induced significant cell growth arrest and apoptosis in ALL cells (p<0.0001) and resulted in up-regulation of P-AMPK (Thr172) and P-Akt (Thr308). The phosphorylation of Akt at Thr308 was mediated by AMPK-dependent activation of IGF-1R/IRS-1, since down-regulation of AMPK using shRNA abolished the activation of both P-Akt (Thr308) and IGF-1R (Tyr1131). Inhibition of AMPK activity with either CC or shRNA sensitized ALL cells to RAS inhibition (p<0.001). In addition, co-targeting RAS pathway signaling plus either IGF-1R or Akt pathways resulted in significant cell growth inhibition and apoptosis in ALL cells (p<0.001), confirming the functional relevance of this feedback loop mechanism. Taken together, our results indicate that activation and inhibition of AMPK induce cell death in ALL cells through alternative signaling mechanisms impinging on the expression of critical cell proliferation pathways. The data presented here support a new role for AMPK as a molecular regulatory switch coordinating cellular survival responses by activating PI3K/Akt/mTOR and RAS/RAF/ERK oncogenic pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2011-605

A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG-asparaginase, and doxorubicin

Acute lymphoblastic leukemia (ALL) remains a major cause of death in children. AMP‐activated protein kinase (AMPK) affects the unfolded protein response (UPR), leading to increased vulnerability to endoplasmic reticulum (ER) stress in ALL cells. In vitro, metformin causes ALL cell death via AMPK‐mediated inhibition of the UPR. It was evaluated whether ER stress could be induced in relapsed ALL through a phase I study investigating the safety and feasibility of metformin in combination with relapse induction chemotherapy.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.

Inhibition of the NEDD8 conjugation pathway induces calcium-dependent compensatory activation of the pro-survival MEK/ERK pathway in acute lymphoblastic leukemia

De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C β2 (PKC-β2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.